PROJECT SUMMARY Clinically available antipsychotics are not effective in the remediation of the negative symptoms and cognitive impairments observed in individuals with schizophrenia and are associated with a number of dose-limiting adverse effects. Thus, there is a critical need to develop novel therapeutic approaches for schizophrenia with broader efficacy and fewer adverse effects. Clinical and preclinical studies indicate that selective modulation of the M1 muscarinic acetylcholine receptor may provide a novel approach for the treatment of the different symptoms in schizophrenia. Mechanistically, selective M1 activation fits well with the glutamate hypothesis, or N-methyl-D-aspartate receptor (NMDAR) hypofunction hypothesis of schizophrenia, as both M1 and the NR1a NMDAR subunits are co-localized at specific postsynaptic sites, and activation of M1 by orthosteric mAChR agonists, and more recently, highly selective M1 allosteric agonists and M1 positive allosteric modulators (PAMs), developed during the previous funding period, potentiate NMDAR currents in hippocampal and medial prefrontal cortical neurons, and enhance cognitive performance in multiple hippocampal- and/or prefrontal cortical-driven models. Importantly, we and others have shown that M1 allosteric agonists display brain region- dependent (M1-expression dependent) pharmacology and suffer many of the problems associated with previous orthosteric M1 agonist programs. Thus, M1 PAMs represent the mechanism by which to address the therapeutic potential of selective M1 activation in man to target the negative and cognitive symptom clusters in schizophrenia, alone or in combination with standard antipsychotics and/or cholinesterase inhibitors. In Project 1 of this application, we will conduct a series of studies to establish the efficacy for our lead M1 PAM VU0467319 relative to potential adverse effects that have limited the development of previous M1 ligands and to establish a functional biomarker strategy to support future clinical dose-finding studies in Project 4 of this application.